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Real-time polymerase chain reaction

in molecular biology, real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction (Q-PCR/qPCR/qrt-PCR) or kinetic polymerase chain reaction (KPCR), is a laboratory technique based on the PCR, which is used to amplify and simultaneously quantify a targeted DNA molecule. It enables both detection and quantification (as absolute number of copies or relative amount when normalized to DNA input or additional normalizing genes) of one or more specific sequences in a DNA sample.

The procedure follows the general principle of polymerase chain reaction; its key feature is that the amplified DNA is detected as the reaction progresses in real time, a new approach compared to standard PCR, where the product of the reaction is detected at its end. Two common methods for detection of products in real-time PCR are: (1) non-specific fluorescent dyes that intercalate with any double-stranded DNA, and (2) sequence-specific DNA probes consisting of oligonucleotides that are labeled with a fluorescent reporter which permits detection only after hybridization of the probe with its complementary DNA target.

Frequently, real-time PCR is combined with reverse transcription to quantify messenger RNA and Non-coding RNA in cells or tissues.

Abbreviations used for real-time PCR methods vary widely and include RTQ-PCR, Q-PCR or qPCR. [1] Real-time reverse-transcription PCR is often denoted as qRT-PCR,[2], RRT-PCR,[3] or RT-rt PCR.[4] The acronym RT-PCR commonly denotes reverse-transcription PCR and not real-time PCR, but not all authors adhere to this convention.[5]

Background

Real time quantitative PCR uses fluorophores in order to detect levels of gene expression. (*)

Cells in all organisms regulate gene expression and turnover of gene transcripts (messenger RNA, abbreviated to mRNA), and the number of copies of an mRNA transcript of a gene in a cell or tissue is determined by the rates of its expression and degradation.

Northern blotting is often used to estimate the expression level of a gene by visualizing the abundance of its mRNA transcript in a sample. In this method, purified RNA is separated by agarose gel electrophoresis, transferred to a solid matrix (such as a nylon membrane), and probed with a specific DNA or RNA probe that is complementary to the gene of interest. Although this technique is still used to assess gene expression, it requires relatively large amounts of RNA and provides only qualitative or semiquantitative information of mRNA levels.

In order to robustly detect and quantify gene expression from small amounts of RNA, amplification of the gene transcript is necessary. The polymerase chain reaction is a common method for amplifying DNA; for mRNA-based PCR the RNA sample is first reverse transcribed to cDNA with reverse transcriptase.

Development of PCR technologies based on reverse transcription and fluorophores permits measurement of DNA amplification during PCR in real time, i.e., the amplified product is measured at each PCR cycle. The data thus generated can be analysed by computer software to calculate relative gene expression in several samples, or mRNA copy number. Real-time PCR can also be applied to the detection and quantification of DNA in samples to determine the presence and abundance of a particular DNA sequence in these samples.

Real-time PCR with double-stranded DNA-binding dyes as reporters

A DNA-binding dye binds to all double-stranded (ds)DNA in PCR, causing fluorescence of the dye. An increase in DNA product during PCR therefore leads to an increase in fluorescence intensity and is measured at each cycle, thus allowing DNA concentrations to be quantified. However, dsDNA dyes such as SYBR Green will bind to all dsDNA PCR products, including nonspecific PCR products (such as Primer dimer). This can potentially interfere with or prevent accurate quantification of the intended target sequence.

1. The reaction is prepared as usual, with the addition of fluorescent dsDNA dye.
2. The reaction is run in a Real-time PCR instrument, and after each cycle, the levels of fluorescence are measured with a detector; the dye only fluoresces when bound to the dsDNA (i.e., the PCR product). With reference to a standard dilution, the dsDNA concentration in the PCR can be determined.

Like other real-time PCR methods, the values obtained do not have absolute units associated with them (i.e., mRNA copies/cell). As described above, a comparison of a measured DNA/RNA sample to a standard dilution will only give a fraction or ratio of the sample relative to the standard, allowing only relative comparisons between different tissues or experimental conditions. To ensure accuracy in the quantification, it is usually necessary to normalize expression of a target gene to a stably expressed gene (see below). This can correct possible differences in RNA quantity or quality across experimental samples.

Fluorescent reporter probe method

Fluorescent reporter probes detect only the DNA containing the probe sequence; therefore, use of the reporter probe significantly increases specificity, and enables quantification even in the presence of non-specific DNA amplification. Fluorescent probes can be used in multiplex assays—for detection of several genes in the same reaction—based on specific probes with different-coloured labels, provided that all targeted genes are amplified with similar efficiency. The specificity of fluorescent reporter probes also prevents interference of measurements caused by primer dimers, which are undesirable potential by-products in PCR. However, fluorescent reporter probes do not prevent the inhibitory effect of the primer dimers, which may depress accumulation of the desired products in the reaction.

The method relies on a DNA-based probe with a fluorescent reporter at one end and a quencher of fluorescence at the opposite end of the probe. The close proximity of the reporter to the quencher prevents detection of its fluorescence; breakdown of the probe by the 5' to 3' exonuclease activity of the Taq polymerase breaks the reporter-quencher proximity and thus allows unquenched emission of fluorescence, which can be detected after excitation with a laser. An increase in the product targeted by the reporter probe at each PCR cycle therefore causes a proportional increase in fluorescence due to the breakdown of the probe and release of the reporter.

1. The PCR is prepared as usual (see PCR), and the reporter probe is added.
2. As the reaction commences, during the annealing stage of the PCR both probe and primers anneal to the DNA target.
3. Polymerisation of a new DNA strand is initiated from the primers, and once the polymerase reaches the probe, its 5'-3'-exonuclease degrades the probe, physically separating the fluorescent reporter from the quencher, resulting in an increase in fluorescence.
4. Fluorescence is detected and measured in the real-time PCR thermocycler, and its geometric increase corresponding to exponential increase of the product is used to determine the threshold cycle (CT) in each reaction.

Real time quantitative PCR using TaqMan^® probes. (1) In intact probes, reporter fluorescence is quenched. (2) Probes and the complementary DNA strand are hybridized and reporter fluorescence is still quenched. (3) During PCR, the probe is degraded by the Taq polymerase and the fluorescent reporter released. (*)

Quantification

Quantifying gene expression by traditional methods presents several problems. Firstly, detection of mRNA on a Northern blot or PCR products on a gel or Southern blot is time-consuming and does not allow precise quantification. Also, over the 20-40 cycles of a typical PCR, the amount of product reaches a plateau determined more by the amount of primers in the reaction mix than by the input template/sample.

Relative concentrations of DNA present during the exponential phase of the reaction are determined by plotting fluorescence against cycle number on a logarithmic scale (so an exponentially increasing quantity will give a straight line). A threshold for detection of fluorescence above background is determined. The cycle at which the fluorescence from a sample crosses the threshold is called the cycle threshold, Ct. The quantity of DNA theoretically doubles every cycle during the exponential phase and relative amounts of DNA can be calculated, e.g. a sample whose Ct is 3 cycles earlier than another's has 23 = 8 times more template. Since all sets of primers don't work equally well, one has to calculate the reaction efficiency first. Thus, by using this as the base and the cycle difference C(t) as the exponent, the precise difference in starting template can be calculated (in previous example, if efficiency was 1.96, then the sample would have 7.53 times more template).

Amounts of RNA or DNA are then determined by comparing the results to a standard curve produced by real-time PCR of serial dilutions (e.g. undiluted, 1:4, 1:16, 1:64) of a known amount of RNA or DNA. As mentioned above, to accurately quantify gene expression, the measured amount of RNA from the gene of interest is divided by the amount of RNA from a housekeeping gene measured in the same sample to normalize for possible variation in the amount and quality of RNA between different samples. This normalization permits accurate comparison of expression of the gene of interest between different samples, provided that the expression of the reference (housekeeping) gene used in the normalization is very similar across all the samples. Choosing a reference gene fulfilling this criterion is therefore of high importance, and often challenging, because only very few genes show equal levels of expression across a range of different conditions or tissues. [6][7]

Real-time PCR can be used to quantify nucleic acids by two strategies - Relative quantification and Absolute quantification. Relative quantification measures the fold-difference (2X, 3X etc.) in the target amount. Absolute quantification gives the exact number of target molecules present by comparing with known standards. The quality of Standard is important for accurate quantification. [8]

Applications of real-time polymerase chain reaction

There are numerous applications for real-time polymerase chain reaction in the laboratory. It is commonly used for both diagnostic and basic research.

Diagnostic real-time PCR is applied to rapidly detect nucleic acids that are diagnostic of, for example, infectious diseases, cancer and genetic abnormalities. The introduction of real-time PCR assays to the clinical microbiology laboratory has significantly improved the diagnosis of infectious diseases, [9] and is deployed as a tool to detect newly emerging diseases, such as flu, in diagnostic tests.[10]

In research settings, real-time PCR is mainly used to provide quantitative measurements of gene transcription. The technology may be used in determining how the genetic expression of a particular gene changes over time, such as in the response of tissue and cell cultures to an administration of a pharmacological agent, progression of cell differentiation, or in response to changes in environmental conditions.

References

1. ^ VanGuilder HD, Vrana KE, Freeman WM (2008). "Twenty-five years of quantitative PCR for gene expression analysis". Biotechniques 44 (5): 619–626. doi:10.2144/000112776. PMID 18474036.
2. ^ Udvardi MK, Czechowski T, Scheible WR (2008). "Eleven Golden Rules of Quantitative RT-PCR". Plant Cell 20 (7): 1736–1737. doi:10.1105/tpc.108.061143. PMID 18664613.
3. ^ Spackman E, Suarez DL (2008). "Type A influenza virus detection and quantitation by real-time RT-PCR". Methods Mol Biol 436: 19–26. doi:10.1007/978-1-59745-279-3_4. PMID 18370037.
4. ^ Gertsch J, Güttinger M, Sticher O, Heilmann J. (2002). "Relative quantification of mRNA levels in Jurkat T cells with RT-real time-PCR (RT-rt-PCR): new possibilities for the screening of anti-inflammatory and cytotoxic compounds". Pharm Res 19: 1236–1243. doi:10.1023/A:1019818814336. PMID 18370037.
5. ^ edited by Julie Logan, Kirstin Edwards, and Nick Saunders. (2009). Logan J, Edwards K, Saunders N. ed. Real-Time PCR: Current Technology and Applications. Caister Academic Press. ISBN 978-1-904455-39-4.
6. ^ Nailis H, Coenye T, Van Nieuwerburgh F, Deforce D, Nelis HJ (2006). "Development and evaluation of different normalization strategies for gene expression studies in Candida albicans biofilms by real-time PCR". BMC Mol Biol. 7: 25. doi:10.1186/1471-2199-7-25. PMID 16889665.
7. ^ Nolan T, Hands RE, Bustin SA (2006). "Quantification of mRNA using real-time RT-PCR.". Nat. Protoc. 1 (3): 1559–1582. doi:10.1038/nprot.2006.236. PMID 17406449.
8. ^ S. Dhanasekaran,T. Mark Doherty, John Kenneth and TB Trials Study Group. (2010 Mar). "Comparison of different standards for real-time PCR-based absolute quantification". Immunol Methods. 354(1-2) (1-2): 34–9. doi:10.1016/j.jim.2010.01.004. PMID 20109462.
9. ^ Sails AD (2009). "Applications in Clinical Microbiology". Real-Time PCR: Current Technology and Applications. Caister Academic Press. ISBN 978-1-904455-39-4.
10. ^ FDA Authorizes Emergency Use of Influenza Medicines, Diagnostic Test in Response to Swine Flu Outbreak in Humans. FDA News, April 27, 2009.